PROJECT SUMMARY/ABSTRACT The central goal of the proposed research is to determine mechanisms by which viral infections lead to loss of tolerance (LOT) to oral antigen and induce celiac disease (CD) and to design strategies to prevent virus- induced LOT and CD onset. Viral infections are increasingly recognized as contributing factors in the pathogenesis of autoimmune inflammatory diseases. However, little is known about the biological features that enable a virus to provoke inflammatory tissue injury. CD is a T cell-mediated intestinal disorder with an autoimmune component characterized by an inflammatory anti-gluten immune response that occurs exclusively in gluten-exposed persons with HLA DQ2 or DQ8 alleles. Approximately 45% of the U.S. population expresses DQ2 or DQ8, yet only 1% of the population develops the disease, indicating that additional factors contribute to disease induction. Reoviruses are human dsRNA viruses recently linked to CD. These viruses infect the murine intestine and can be genetically manipulated to identify viral determinants of autoimmune host responses. We discovered that reovirus strain T1L abrogates tolerance to dietary antigens including gluten using a pathway dependent on IRF1, whereas strain T3D-RV does not. Induction of protective immunity to T1L occurs by a mechanism distinct from that required for immunopathology. We propose to enhance an understanding of how viruses influence the development of autoimmune inflammatory disorders and prevent CD by identifying virus and host factors that influence LOT to dietary antigen and defining strategies to block virus-induced LOT and CD development. To achieve this goal, we have designed three well-integrated specific aims. In Specific Aim 1, we will define the viral determinants and associated host pathways responsible for LOT using T1L x T3D-RV reassortant viruses and engineered viral mutants to define functional domains of protein products responsible for LOT. We will elucidate mechanisms underlying IRF1-mediated TH1 immunity following reovirus infection using mice lacking IRF1 or IRF1-inducing factors in specific cell types. In Specific Aim 2, we will define the effect of T1L and T3D-RV infection on the composition of microbial communities. We will also determine the contribution of the intestinal microbiota to reovirus-induced LOT by assessing LOT in antibiotic-treated and germ-free mice with and without defined microbiota. In Specific Aim 3, we will define the temporal relationship of T1L infection and gluten introduction on LOT using mice engineered to express the human DQ8 risk allele. We will test whether inoculation with inactivated or attenuated reovirus prior to gluten introduction prevents LOT mediated by T1L. This project extends a productive collaboration of internationally recognized PIs with complementary expertise in CD, mucosal immunology, and virology. Knowledge gained through these efforts will enhance an understanding of how viral infections lead to the development of gastrointestinal inflammatory disorders and foster new strategies to prevent CD in genetically vulnerable individuals.